1. Technical Field
The disclosure relates to an imaging apparatus, a microscope system, and an imaging method for imaging an object to acquire an image.
2. Related Art
In recent years, an electronic imaging technique has progressed in the technical field of microscopic observation, and various imaging methods and image processing methods have been developed appropriately for an observation target. As an example, there has been proposed a system which displays a single image with a wide range and high definition by stitching a plurality of images obtained by imaging a plurality of regions within an object as an observation target. Such a system is also called a virtual slide system. For example, Japanese Laid-open Patent Publication No. 2008-191427 discloses a technique for dividing an observed region of a living tissue into small sections and connecting images acquired by imaging in each of the small sections.
In such an imaging technique, the number of times of performing imaging increases depending on the number of small sections. Thus, there is a need to increase the speed of an imaging operation. The increase in the imaging speed involves how to increase the speed of autofocus when imaging the small sections.
There has also been developed a technique for three-dimensionally displaying an object by extracting a confocal point from a plurality of images of the object observed with a microscope. Generally, in a confocal microscopy, it takes a long time to acquire image data since an optical system such as an objective lens should be operated in the optical axis direction (Z direction) in order to change a confocal plane.
In view of such a situation, Japanese Laid-open Patent Publication No. 11-211439 discloses a technique of focus-free imaging by moving a stage with tiling a focal plane on an object with respect to a moving direction (e.g., X direction) of the stage and performing imaging with changing the surface position of the object in the Z-axis direction. This technique can detect the object included in the thickness corresponding to the tilt angle of the focal plane, and thus eliminate the necessity of the operation in the Z direction.
In order to speed up the imaging operation in the virtual slide system, it would appear that a confocal imaging technique disclosed in Japanese Laid-open Patent Publication No. 11-211439 can be applied to the system disclosed in Japanese Laid-open Patent Publication No. 2008-191427. With this combination, the autofocus may not be required with respect to the small sections, thereby speeding up the imaging of the whole observed region.
However, a trade-off arises between a moving velocity on an object plane and a range of detectable thickness determined according to a tilt angle of a focal plane. With respect to an imaging apparatus capable of imaging an area of 200 μm wide at one time, for example, a case where a focal plane PFC is tilted by 10 μm in a thickness direction as shown in FIG. 18 is compared to a case where the focal plane PFC is tilted by 4 μm in the thickness direction as shown in FIG. 19. Each of imaging areas C1, C2, . . . shown in FIGS. 18 and 19 indicates an area on an object OB where image information can be acquired by a single imaging operation. In FIGS. 18 and 19, a scale size in a vertical direction is larger than that in a horizontal direction.
In FIG. 18, image information of 10 μm in the thickness direction can be acquired in a single imaging operation. In other words, the focal plane in the object OB can be searched within a range of 10 μm. In FIG. 19, on the other hand, only the image information of 4 μm in the thickness direction can be acquired. However, in tilt and image formation per 1 μm, for example, the imaging is performed by moving the stage by about 20 μm at a time in the case shown in FIG. 18 while the imaging can be performed by moving the stage by 50 μm at a time in the case shown in FIG. 19. Therefore, in the latter case, it is possible to take an image of the whole object OB with the smaller number of times of performing the imaging, i.e., in a short time.
For this reason, a smaller amount of tilt of the object is desirable in order to shorten the time of imaging the whole observation target. To meet this condition, however, it is necessary for the observation target to be located within a range in the thickness gave from the focal plane tilting.
In observing an object sandwiched between parallel plates, such as a prepared slide having a biological sample placed on a glass slide and sealed with a coverslip, the object is generally placed to be substantially orthogonal to an optical axis. In observing the object using the tilt and image formation described above, when the tilt amount of the focal plane increases, blurring on a non-focused region may be unnatural. Therefore, it is preferable to set the tilt amount appropriately to be controlled within a specified range.
Thus, in observing the object using the tilt and image formation, a suitable tilt amount of the focal plane for observation is set under various conditions, while the object plane needs to be in a Z-direction range defined according to the tilt amount of the focal plane.
When an object as an observation target is a biological sample, a prepared slide in many cases is not completely orthogonal to an optical axis due to an uneven thickness or the like of the biological sample. Therefore, the tilt of the prepared slide affects imaging significantly at a level of depth of focus. Consequently, auto-focusing is usually performed each time the observation region on the object changes. By comparison, the above-described observation with the tilt and image formation is a technique that does not require auto-focusing. It is, however, difficult to achieve both a Z-direction imaging range for compensating the tilted prepared slide or the above-described uneven thickness, and a throughput.